The present invention relates to an improved process for the preparation of stable bacosites enriched fraction in non-hygroscopic form from the herb Bacopa monniera. 
Plant based drugs and formulations are showing a rising trend globally for the health care due to the biosafe attributes they possess over modern synthetic medicines. Amongst the numerous traditional Indian medicinal herbs, standardized extract of Bacopa monniera known as xe2x80x98Brahmixe2x80x99 in Hindi is finding increasing use as a constituent of modern herbal nutraceuticals aimed at supporting brain and nerve function and enhancing memory, alertness and mental concentration (Ray Sahelian, M.D. xe2x80x9cMind Boostersxe2x80x9d, 1999, Amazon Publishing House, USA).
Bacopa monniera has been used in India since ancient times in Ayurvedic preparations as a brain and a nerve tonic (Chunekar K C, xe2x80x9cBhav Prakasha Nighantuxe2x80x9d, Hindi translation, Varanasi, 1960: 372). In a clinical trial carried out at the Banaras Hindu University. (Singh R H and Singh Lallan, xe2x80x9cStudies on the Anti-anxiety effect of the Medhya Rasayana Drug, Brahmi Bacopa monniera Wettst. Part 1, Journal of Res. Ayurveda and Sidha Vol 1: 133-148, 1980). Bacopa monniera in the form of brahmi syrup when administered to 35 patients suffering from anxiety neurosis, it was concluded that 4 weeks of treatment with brahmi significantly reduced the level of anxiety amongst the patients with improvement in the mental performance and memory of the treated patients. The beneficial effects of Bacopa monniera on the intelligence and mental performance were further investigated at Banaras Hindu University in a trial carried out on 20 school children over a period of three months. (Sharma R, Chaturvedi C, Tewari P V, xe2x80x9cEfficacy of Bacopa monniera in revitalizing intellectual functions in Childrenxe2x80x9d Journal Res. Edu, Ind Med, Jan-Jun.: 1-12, 1987).
Systematic Chemical Examination of Bacopa monniera was first reported by N Chatterjee, R P Rastogi and M L Dhar, (Indian Journal of Chemistry, Vol 1, May 1963). They reported the occurrence of two saponins designated as Bacoside A and B which are present in a concentration of over 2% in the dry plant. The molecular structure of the bacosides A and B was subsequently elucidated. (Chatterjee N, Rastogi R P and Dhar M L, Indian J Chem, Vol 3: 24, 1965 and Basu N, Rastogi P and Dhar M L, Indian J Chem Vol 5: 84, 1967). These studies also reported the physical properties of the bacosides AandB:
An analytical method based on high performance thin layer chromatography (HPTLC), for the determination of bacoside-A content in Bacopa monniera has been reported by Gupta et al (A P Gupta, S Mathur, M M Gupta and Sushil Kumar, xe2x80x9cEffect of the method of drying on the bacoside-A content of the harvested Bacopa monniera shoots revealed using a high performance thin layer chromatography methodxe2x80x9d, Journal of Medicinal and Aromatic Plants Vol, 20: 1052-1055, 1998). The bioactivity of Bacopa monniera extract has been studied by evaluating the avoidance responses in Rats in an extensive trial carried out at the Central Drug Research Institute, Lucknow, India (H K Singh, R P Rastogi, R C Srimal and B N Dhawan, Phytotherapy Research, Vol 2 (2): 70-75, 1988). It has been concluded in this study that Bacosides A and B are the active constituents of Bacopa monniera which are responsible for the enhanced mental performance and retention capacity.
The authentication of the traditional claims of brahmi was investigated at the Central Drug Research Institute by studying the effect of alcoholic extract of this plant on acquisition, consolidation and retention of the three memory related behavioral responses in albino rats. (Singh H K and Dhawan B N, xe2x80x9cNeuropsychopharmacological effects of the Ayurvedic nootropic Bacopa monniera Linn (Brahmi)xe2x80x9d, Indian Journal of Pharmacology, Vol 29 (5): S359-S365, 1997). In this study, bacosides were also found to be safe in regulatory, pharmacological and toxicological studies carried out on normal healthy male volunteers. This study was designated as Phase-I clinical trial. Bacopa extract was administered to human volunteers for 4 weeks in single and multiple doses in double blind placebo controlled and non-crossover regulatory clinical trial. The mechanism of action of the facilitatory effect of bacosides was attributed to their enhanced protein kinase activity and production of an increased level of protein in hippocampus. Another conclusion of this study was that the bacosides attenuated the retrograde amnesia produced by immobilization induced stress and scopolamine. P A Thakurdesai, P L Kole and A. N Nagappa (www.Pharmabiz.com/newsfeat/feat/112.com) have stressed the necessity of using standardized extracts for achieving desired efficacy.
Considering the great importance and potential of bacosides, derived from Bacopa monniera, in human health, the information about an efficient process suitable for industrial production is not available in the literature. Patent literature also does not disclose any information on the subject of efficient production technology of bacosides. The literature contains a few lab scale methods for the preparation of an extract of Bacopa monniera containing bacosides. The method of isolation adopted by Chatterjee et al (N Chatterjee, R. P. Rastogi and M. L. Dhar, Indian Journal of Chemistry, Vol 1, May 1963; Chatterjee N, Rastogi R P and Dhar M L, Indian J Chem, Vol 3: 24, 1965) and further modified by Singh et al (H K Singh, R P Rastogi, R C Srimal and B N Dhawan, Phytotherapy Research, Vol 2 (2): 70-75, 1988), describes the extraction of the Bacopa monniera dried herb with alcohol, wherein the dried herb material is first moistened with water and then extracted with alcohol, which is concentrated under reduced pressure and repeatedly macerated with benzene for defatting. The filtrate is diluted to 60% concentration of alcohol and is treated with an excess of lead acetate. The lead salts are filtered and the residual lead was removed from the filtrate with hydrogen sulphide. The pH of the filtrate is adjusted to 6.4 with sodium carbonate and concentrated at 50xc2x0 C. under vacuum to one third of its volume and partitioned repeatedly with butanol and water. The butanol fraction on concentration under vacuum deposits a powder containing bacosides AandB. The powder is then crystallized from alcohol as colourless needles. An additional amount of bacosides is obtained from the filtrate by freeing it from solvent and macerating the residue with acetone. This method apart from being very tedious and time consuming involves the use of benzene (potentially carcinogenic) and lead salt (highly poisonous). Although the process attempts to remove the residual lead with hydrogen sulphide gas, the complete removal is doubtful in view of involvement of gas-liquid mass transfer where high efficiencies are difficult to achieve. The presence of these toxic chemicals even in traces will make the quality of the final product questionable. This method is not suited for scaling up to industrial production due to being tedious and health hazardous. In the method adopted for the study carried out by H K Singh and B N Dhawan (Journal of Ethanopharmacology, Vol 5: 205-214, 1982), to evaluate the effect of brahmi extract on avoidance responses in rats, the air dried plant material is extracted with 90% ethanol by soxhlet extraction apparatus and the extract obtained is mixed with 10% gum acacia for feeding the rats. In this study no attempt was made to monitor the bacosides contents of the extracts. In an another study carried out at the University of Madras on the anti cancer activity of Bacopa monniera (V. Elangovan, S Govindasamy, N Ramamoorthy and K. Balasubramanian, xe2x80x9cIn vitro studies on the anticancer activity of Bacopa monniera, Fitoterapia, Vol LXVI (3): 211-215, 1995), the procedure for the extraction of bacosides consists of soaking the powdered plant material in 95% ethanol for 48 hr, concentrating the extract under vacuum and drying it by lypholisation. No attempt was made to measure the bacoside concentration of the extract. Standardised extract of Bacopa monniera containing 20-30% bacosides AandB is being manufactured and marketed by a few commercial firms, but the process of production is not disclosed. (Product Information Brochure of M/s Sabinsa Corporation, 121, Ethel Road West, Piscataway, N.J. 08854, USA and M/s Himalaya USA, M/s Velvette International, Chennai, India, M/s Dalmia Industries Ltd, India).
A major problem encountered during the process was the difficulty of obtaining the stable final product in the form of a dry free flowing powder as the active constituents (bacosides) are highly hygroscopic.
The main object of the present invention is to provide an improved process for the preparation of bacosites enriched fraction in non-hygroscopic form from the herb Bacopa monniera overcoming the disadvantages of the hitherto known processes.
Another object of the present invention is to provide an improved process for preparation of bacosites enriched fraction from Bacopa monniera using relatively non toxic solvents like hexane, acetone and methanol.
Yet another object of the present invention is to provide an improved process for removing unwanted constituents of Bacopa monniera herb by extracting the defatted herb with acetone.
Still another object of the present invention is to provide an improved process for stabilization of bacoside containing extract by addition of non-toxic stabilizing agents such as mannitol, maltodextrin, xcex2-cyclodextrin or polyvinyl alcohol.
One more object of the present invention is to provide an improved process for purifying the Bacopa monniera extract using a countercurrent xe2x80x98KARRxe2x80x99 liquidxe2x80x94liquid extraction column fitted with a reciprocating agitator.
Accordingly, the present invention provides an efficient process for the preparation of stable bacosites enriched fraction in non-hygroscopic form from the herb Bacopa monniera.